Thymosin beta 4 (T beta 4; also TB4X and Fx) is a 5.0 kDa member of the beta-thymosin family of molecules. It Plays an important role in the organization of the cytoskeleton. It is reported that TMSB4X can combinant with PFN1 to inhibit actin polymerization. A binding ELISA assay was conducted to detect the interaction of recombinant human TMSB4X and recombinant human PFN1. Briefly, TMSB4X were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to PFN1-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-TMSB4X pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50μL stop solution to the wells and read at 450nm immediately. The binding activity of TMSB4X and PFN1 was shown in Figure 1, the EC50?for this effect is 2.98 ng/mL.